0361-9230/90 $3.00 + .OO
Brain Research Bulletin, Vol. 24, pp. 509-516. Q Pergamon Press plc, 1990. Printed in the U.S.A.
Medioventral Medulla-Induced
Locomotion
N. KINJO, Y. ATSUTA, M. WEBBER, R. KYLE, R. D. SKINNER AND E. GARCIA-RILL Department
of Anatomy,
University of Arkansas for Medical Sciences,
Little Rock, AR 72205
Received 16 October 1989
KINJO, N., Y. ATSUTA, M. WEBBER, R. KYLE, R. D. SKINNER AND E. GARCIA-RILL. Medioventral medulla-induced locomotion. BRAIN RES BULL 24(3) 509-516, 1990. -Previous anatomical studies demonstrated the presence of descending projections from the physiologically identified mesencephalic locomotor region (MLR) to the medioventml medulla (MED) in the cat. The present experiments were designed to determine if a similar low threshold locomotion-inducing area is present in the rat medulla. In addition, the nature of the neurochemical control of this area of the brain was explored using localized injections of neurochemical agents in the decembrate rat during locomotion on a treadmill. A region virtually identical to that reported in the cat was found to lead
to controlled locomotion on a treadmill following stimulation at low amplitude currents (560 p,A). Injections of cholinergic agonists into the MED of the rat induced locomotion which could be blocked by injections of cholinergic antagonists. In addition, injections of GABA antagonists were found to induce stepping which could be blocked by injections of GABA or GABA agonists. Substance P (SP) also was found to induce walking following injection into the MED of the rat. Injections of an excitatory amino acid agonist (NMDA) also were found to induce locomotion in the rat. These effects were blocked by injections of an excitatory amino acid antagonist (APV). Since these results had not been reported for the cat MED, a short series of experiments revealed that the MBD in the cat also responded to NMDA. Locomotion
Mesencephalic
locomotor
region
NMDA
the presence of locomotion-inducing sites in the vicinity of cholinergic neurons of the pedunculopontine nucleus (PPN) (7). The suggestion that at least some of the descending efferents of the MLR to the MED are cholinergic was supported by results showing induction of locomotion following localized injections of cholinergic agonists into the MED of the cat (9). Locomotion, however, was induced for only short periods, leading to. the speculation that the cholinergic input to the MED merely triggers locomotion. The implicit assumption in this suggestion is that other transmitter systems also may control the locomotion-related output of the MED. Indeed, substance P (SP) and GABAergic agents also were found to influence the MED of the cat. Herein are described results in the rat suggesting that: 1) low amplitude electrical stimulation of the MED can induce stepping, 2) localized injections of neuroactive substances into the MED also can induce locomotion, 3) cholinergic, GABAergic, SP and excitatory amino acid inputs and/or intemeurons influence the MED of the rat and 4) the effects of excitatory amino acid agonists and antagonists, previously not reported for the cat MED, were confiied to have similar effects in that species. Preliminary results have been reported (17).
ELECTRICAL stimulation of a region of the caudal midbrain was reported to induce locomotion on a treadmill in the precolliculartransected cat and was termed the mesencephalic locomotor region (MLR) (32). Stimulation of the same region has been found to induce progressive movements in intact cats (40). Presumably, descending projections of this region influence spinal locomotor pattern generators in order to induce stepping. In anterograde labeling studies, it has been shown that the MLR projects to the dorsolateral pons and medulla, and to the medioventral medulla (MED) (13). The former projection was ascribed to labeling of descending trigeminal elements, whereas the later projections were considered to be from the MLR itself (6,13). Subsequent anatomical studies confirmed these findings (39), which were supported by additional results describing blockade of MLRinduced locomotion by cooling in the area of the MED (31). Definitive experiments using localized, low amplitude electrical and low concentration chemical stimulation of the MED established the presence of this locomotion-inducing area in the cat brain (9). In addition, a significant number of single cells recorded in this area were found to respond orthodromically at short latency to MLR stimulation and to respond antidromically following stimulation of the spinal cord, i.e., the MED contains reticulospinal neurons (10). The present experiments were designed to determine the degree to which the MED is involved in locomotion in the rat. Previous studies had established the existence of the MLR in the rat (35), and found mat a large number of neurons in the area of the rat MLR were retrogradely labeled following injections of retrograde tracers into the area of the MED (12). A series of studies using microstimulation and immunocytochemical techniques established
METHOD
Surgery Forty-five adult Sprague-Dawley rats were anesthetized with halothane during surgical procedures. The surgical procedures, methods of recording electromyograms (EMG) and other techniques are described in a companion article (8). It should be noted that there is no spontaneous locomotion in the precollicular-
509
510
transected preparation and stepping must be induced electrically or chemically. Electrically Induced Locomotion In the initial studies for localizing the MED, a semimicroelectrode (resistance 200-300 Kohm) was introduced stereotaxically and stimuli applied throughout the medial medullary reticular formation. For chemically induced locomotion, a stainless steel tubing cannula (o.d. 250 pm) with an internal diameter which snuggly accommodated a 100~Frn diameter wire made up the cannultielectrode ensemble. The wire (resistance 70-100 Kohm) was insulated except at the cut end and protruded from the tip of the cannula by 0.5 mm. The semimicroelectrode or the cannula/ electrode ensemble was lowered stereotaxically (coordinates P 1.O-3.0, L O-2.0) in 0.25mm steps from an initial position 3-4 mm dorsal to the MED. Monopolar electrical stimulation was applied (reference electrode in neck muscles) at a frequency of 5-60 Hz using 0.5-l.O-msec duration pulses. The criteria for determining the presence of a locomotion-inducing site consisted of, 1) low threshold- (560 PA) induced alternation of both forelimbs, both hindlimbs or all four limbs, 2) increasing current amplitude-induced changes in the step cycle (from a walk to a trot to a gallop), and 3) locomotion ceased upon termination of stimulation. Electromyograms (EMG), heart rate and stimulus pulses were recorded on FM tape and locomotion was videotaped. See the companion article for additional details (8).
KINJO ET AL.
underestimate the actual spread. The sections then were incubated for NADPH diaphorase histochemical labeling of cholinergic PPN neurons (1144). Sufficient background labeling of the pons and medulla was present in these sections, allowing assessment of the locations of various landmarks and the MED stimulation/injection site even though there were no NADPH diaphorase positive neurons in the area of the MED. In some cases (n = 8) the sections were stained instead with cresyl violet to determine cytoarchitectonic boundaries. In other cases (n=8) 0.05 ~.l of Fluoro-Gold was injected bilaterally into the cervical enlargement (C,) one week prior to electrophysiological recordings. The locations of the cannula/electrode ensemble then were determined in relation to the locations of Fluoro-Gold labeled medullary reticulospinal neurons. Studies in Cat While the present results describe findings in the rat MED, some of which previously had been reported in the cat (9), the effects of excitatory amino acid agonists and antagonists described herein for the rat had not been reported for the cat. For that reason, a series of experiments (n=6) was carried out in precollicularpostmammillary-transected cats to determine if these agents also influenced MED activity in that species. The methods used in this series in the cat were similar to those previously reported (9), and included histological reconstruction using NADPH diaphorase histochemistry (11). RESULTS
Drug Injections
Electrically Induced Locomotion
The cannula tubing was cut at various lengths yielding known internal volumes (0.3 ~1, 0.5 pl and 0.7 ~1). Compounds to be injected were mixed in distilled water and pH usually was maintained at 7.2-7.4. The rate of injection invariably was 1 p..Ymin. The following compounds were used: GABAergic agentsGABA (0. l-l M), muscimol (MUS) (l-5 mM), picrotoxin (PIC) (l-5 mM), bicuculline (BIC) (l-5 mM). Substance P (SP) (l-5 mM). Excitatory amino acid agents-glutamic acid (GLU) (0. l-l M), aspartic acid (ASP) (0.1-l M), dihydrokainic acid (DHK) (5-10 n&l), n-methyl-d-aspartic acid (NMDA) (l-20 mM), aminophosphonovalerionic acid (APV-5) (l-5 mM). Cholinergic agents-carbachol (CAR) (lo-50 mM), methacholine (l-5 mM), arecoline (0. l-l mM), atropine (0. l-l mM). Once a low threshold locomotion-inducing site had been localized, the stimulating wire was removed and the appropriate volume of drug used to fill the cannula. At least 15 min elapsed between electrical testing and drug application, as well as between drug applications. The same parameters and techniques used in the companion studies on the MLR (8) were used in order to assess drug-induced effects in the MED.
It should be stressed that only the medial areas of the pontomedullary junction were probed for locomotion-specific sites. Only sites which when activated at low current amplitude (560 p.A) led to controlled locomotion involving two or four limbs were considered to be “locomotion-inducing. ” Rhythmic stepping with only one limb was not considered an appropriate effect although it was observed occasionally following activation of the pyramid and along the midline. Stimulation at higher current amplitudes (60-120 WA) was found to induce stepping when applied to several sites throughout the dorsal reticular formation. However, these sites did not yield controlled locomotion and/or did not produce the same effect upon repeated stimulation. Figure 1 shows the distribution of locomotion-inducing stimulation sites throughout the medial reticular formation. Low threshold locomotion-inducing sites were clustered around a region which extended rostrocaudally from the caudal edge of the trapezoid body to the rostra1 edge of the inferior olive. Dorsoventrally, the area extended for about 1 mm dorsal to the pyramid. Mediolaterally, most sites were located around 0.5 mm lateral to the midline. On the other hand, locomotion-inducing sites outside of these boundaries were observed in some animals. For example, low threshold locomotion could be induced in some cases following stimulation along the midline dorsal to the MED. Also, in a few cases, stimulation of an area dorsal to the MED and 0.5-l .O mm lateral to the midline led to locomotion. These locations produced locomotion in only a few cases, however, when evident, stimulation failed to produce replicable stepping. Examination of the histological material revealed that the above distribution of locomotion-inducing sites, the region herein termed the MED, appeared to be located in and around the area of the nucleus reticularis magnocellularis pars beta (23). Some sites were found to be located more ventrally in the area of the nucleus reticularis magnocellularis pars alpha. Other sites were found slightly more dorsally in the ventral part of the nucleus reticularis gigantocellularis. Locomotion was not evident following stimula-
Injection Sites Only one penetration per MED was made. At the end of each experiment, an appropriate volume of Fast Green (0.30.7 ~1) was injected through the cannula at 1 kl/min. Most animals (n = 29) were sacrificed with an overdose of barbiturate and perfused transcardially with fixative and sucrose for NADPH diaphorase histochemistry (44). Frozen sections were cut (40 km) sagittally and the extent of Fast Green spread assessed by overhead projection of wet sections. Drawings of the extent of the Fast Green spread from wet sections later were superimposed on drawings of the same sections stained for NADPH diaphorase. Because the incubation needed for such labeling tends to dissolve the Fast Green except in spots, use of dried sections would tend to
MEDIOVENTRAL
MEDULLA-INDUCED
511
LOCOMOTION
tion. Stepping typically was elicited after 5-20 set of stimulation as the current amplitude was increased to threshold. Locomotion could be maintained for prolonged periods (up to 5 min). As the current amplitude was increased, the step cycle became increasingly rigid leading to “goose-stepping,” especially in the forelimbs. With increasing current such alternation quickly shifted to a trot and a gallop, leading to rigidity beyond 60-80 kA. The threshold for inducing locomotion following stimulation of the MED was 23.62 13.1 p,A (mean and S.D.). Stimulation of the MED did not require high frequencies to maintain stepping. Unlike the MLR, which requires pulse frequencies of 20-60 Hz, the MED could be activated using frequencies as low as 5 Hz. Similar characteristics were reported for the MED in the cat (9). The present experiments did not attempt to survey the entire medulla for locomotion-inducing sites. Areas possibly equivalent to the pontobulbar locomotor strip (PLS) (33), and the dorsal and ventral tegmental fields (21), were not tested in the rat. Rather, this study was limited to the area of the MED found to lead to labeling of MLR neurons after injections of retrograde tracers ( 12). Chemically Induced Locomotion
PIG. 1. (A) Sag&al view of the rat brainstem (midbrain-pons-medulla) at lateral level L 0.4 showing the locations of the central gray (CG), inferior olive (IO), pyramidal tract (PT), nucleus of the sixth cranial nerve (VI) and the level of transection (T). The vertical lines indicate several representative rostrocaudal stereotaxic penetrations using a semimicroelectrode and the effects obtained following stimulation at various depths. The stars denote low threshold- (
Brain Research Bulletin, Vol. 24, pp. 509-516. Q Pergamon Press plc, 1990. Printed in the U.S.A.
Medioventral Medulla-Induced
Locomotion
N. KINJO, Y. ATSUTA, M. WEBBER, R. KYLE, R. D. SKINNER AND E. GARCIA-RILL Department
of Anatomy,
University of Arkansas for Medical Sciences,
Little Rock, AR 72205
Received 16 October 1989
KINJO, N., Y. ATSUTA, M. WEBBER, R. KYLE, R. D. SKINNER AND E. GARCIA-RILL. Medioventral medulla-induced locomotion. BRAIN RES BULL 24(3) 509-516, 1990. -Previous anatomical studies demonstrated the presence of descending projections from the physiologically identified mesencephalic locomotor region (MLR) to the medioventml medulla (MED) in the cat. The present experiments were designed to determine if a similar low threshold locomotion-inducing area is present in the rat medulla. In addition, the nature of the neurochemical control of this area of the brain was explored using localized injections of neurochemical agents in the decembrate rat during locomotion on a treadmill. A region virtually identical to that reported in the cat was found to lead
to controlled locomotion on a treadmill following stimulation at low amplitude currents (560 p,A). Injections of cholinergic agonists into the MED of the rat induced locomotion which could be blocked by injections of cholinergic antagonists. In addition, injections of GABA antagonists were found to induce stepping which could be blocked by injections of GABA or GABA agonists. Substance P (SP) also was found to induce walking following injection into the MED of the rat. Injections of an excitatory amino acid agonist (NMDA) also were found to induce locomotion in the rat. These effects were blocked by injections of an excitatory amino acid antagonist (APV). Since these results had not been reported for the cat MED, a short series of experiments revealed that the MBD in the cat also responded to NMDA. Locomotion
Mesencephalic
locomotor
region
NMDA
the presence of locomotion-inducing sites in the vicinity of cholinergic neurons of the pedunculopontine nucleus (PPN) (7). The suggestion that at least some of the descending efferents of the MLR to the MED are cholinergic was supported by results showing induction of locomotion following localized injections of cholinergic agonists into the MED of the cat (9). Locomotion, however, was induced for only short periods, leading to. the speculation that the cholinergic input to the MED merely triggers locomotion. The implicit assumption in this suggestion is that other transmitter systems also may control the locomotion-related output of the MED. Indeed, substance P (SP) and GABAergic agents also were found to influence the MED of the cat. Herein are described results in the rat suggesting that: 1) low amplitude electrical stimulation of the MED can induce stepping, 2) localized injections of neuroactive substances into the MED also can induce locomotion, 3) cholinergic, GABAergic, SP and excitatory amino acid inputs and/or intemeurons influence the MED of the rat and 4) the effects of excitatory amino acid agonists and antagonists, previously not reported for the cat MED, were confiied to have similar effects in that species. Preliminary results have been reported (17).
ELECTRICAL stimulation of a region of the caudal midbrain was reported to induce locomotion on a treadmill in the precolliculartransected cat and was termed the mesencephalic locomotor region (MLR) (32). Stimulation of the same region has been found to induce progressive movements in intact cats (40). Presumably, descending projections of this region influence spinal locomotor pattern generators in order to induce stepping. In anterograde labeling studies, it has been shown that the MLR projects to the dorsolateral pons and medulla, and to the medioventral medulla (MED) (13). The former projection was ascribed to labeling of descending trigeminal elements, whereas the later projections were considered to be from the MLR itself (6,13). Subsequent anatomical studies confirmed these findings (39), which were supported by additional results describing blockade of MLRinduced locomotion by cooling in the area of the MED (31). Definitive experiments using localized, low amplitude electrical and low concentration chemical stimulation of the MED established the presence of this locomotion-inducing area in the cat brain (9). In addition, a significant number of single cells recorded in this area were found to respond orthodromically at short latency to MLR stimulation and to respond antidromically following stimulation of the spinal cord, i.e., the MED contains reticulospinal neurons (10). The present experiments were designed to determine the degree to which the MED is involved in locomotion in the rat. Previous studies had established the existence of the MLR in the rat (35), and found mat a large number of neurons in the area of the rat MLR were retrogradely labeled following injections of retrograde tracers into the area of the MED (12). A series of studies using microstimulation and immunocytochemical techniques established
METHOD
Surgery Forty-five adult Sprague-Dawley rats were anesthetized with halothane during surgical procedures. The surgical procedures, methods of recording electromyograms (EMG) and other techniques are described in a companion article (8). It should be noted that there is no spontaneous locomotion in the precollicular-
509
510
transected preparation and stepping must be induced electrically or chemically. Electrically Induced Locomotion In the initial studies for localizing the MED, a semimicroelectrode (resistance 200-300 Kohm) was introduced stereotaxically and stimuli applied throughout the medial medullary reticular formation. For chemically induced locomotion, a stainless steel tubing cannula (o.d. 250 pm) with an internal diameter which snuggly accommodated a 100~Frn diameter wire made up the cannultielectrode ensemble. The wire (resistance 70-100 Kohm) was insulated except at the cut end and protruded from the tip of the cannula by 0.5 mm. The semimicroelectrode or the cannula/ electrode ensemble was lowered stereotaxically (coordinates P 1.O-3.0, L O-2.0) in 0.25mm steps from an initial position 3-4 mm dorsal to the MED. Monopolar electrical stimulation was applied (reference electrode in neck muscles) at a frequency of 5-60 Hz using 0.5-l.O-msec duration pulses. The criteria for determining the presence of a locomotion-inducing site consisted of, 1) low threshold- (560 PA) induced alternation of both forelimbs, both hindlimbs or all four limbs, 2) increasing current amplitude-induced changes in the step cycle (from a walk to a trot to a gallop), and 3) locomotion ceased upon termination of stimulation. Electromyograms (EMG), heart rate and stimulus pulses were recorded on FM tape and locomotion was videotaped. See the companion article for additional details (8).
KINJO ET AL.
underestimate the actual spread. The sections then were incubated for NADPH diaphorase histochemical labeling of cholinergic PPN neurons (1144). Sufficient background labeling of the pons and medulla was present in these sections, allowing assessment of the locations of various landmarks and the MED stimulation/injection site even though there were no NADPH diaphorase positive neurons in the area of the MED. In some cases (n = 8) the sections were stained instead with cresyl violet to determine cytoarchitectonic boundaries. In other cases (n=8) 0.05 ~.l of Fluoro-Gold was injected bilaterally into the cervical enlargement (C,) one week prior to electrophysiological recordings. The locations of the cannula/electrode ensemble then were determined in relation to the locations of Fluoro-Gold labeled medullary reticulospinal neurons. Studies in Cat While the present results describe findings in the rat MED, some of which previously had been reported in the cat (9), the effects of excitatory amino acid agonists and antagonists described herein for the rat had not been reported for the cat. For that reason, a series of experiments (n=6) was carried out in precollicularpostmammillary-transected cats to determine if these agents also influenced MED activity in that species. The methods used in this series in the cat were similar to those previously reported (9), and included histological reconstruction using NADPH diaphorase histochemistry (11). RESULTS
Drug Injections
Electrically Induced Locomotion
The cannula tubing was cut at various lengths yielding known internal volumes (0.3 ~1, 0.5 pl and 0.7 ~1). Compounds to be injected were mixed in distilled water and pH usually was maintained at 7.2-7.4. The rate of injection invariably was 1 p..Ymin. The following compounds were used: GABAergic agentsGABA (0. l-l M), muscimol (MUS) (l-5 mM), picrotoxin (PIC) (l-5 mM), bicuculline (BIC) (l-5 mM). Substance P (SP) (l-5 mM). Excitatory amino acid agents-glutamic acid (GLU) (0. l-l M), aspartic acid (ASP) (0.1-l M), dihydrokainic acid (DHK) (5-10 n&l), n-methyl-d-aspartic acid (NMDA) (l-20 mM), aminophosphonovalerionic acid (APV-5) (l-5 mM). Cholinergic agents-carbachol (CAR) (lo-50 mM), methacholine (l-5 mM), arecoline (0. l-l mM), atropine (0. l-l mM). Once a low threshold locomotion-inducing site had been localized, the stimulating wire was removed and the appropriate volume of drug used to fill the cannula. At least 15 min elapsed between electrical testing and drug application, as well as between drug applications. The same parameters and techniques used in the companion studies on the MLR (8) were used in order to assess drug-induced effects in the MED.
It should be stressed that only the medial areas of the pontomedullary junction were probed for locomotion-specific sites. Only sites which when activated at low current amplitude (560 p.A) led to controlled locomotion involving two or four limbs were considered to be “locomotion-inducing. ” Rhythmic stepping with only one limb was not considered an appropriate effect although it was observed occasionally following activation of the pyramid and along the midline. Stimulation at higher current amplitudes (60-120 WA) was found to induce stepping when applied to several sites throughout the dorsal reticular formation. However, these sites did not yield controlled locomotion and/or did not produce the same effect upon repeated stimulation. Figure 1 shows the distribution of locomotion-inducing stimulation sites throughout the medial reticular formation. Low threshold locomotion-inducing sites were clustered around a region which extended rostrocaudally from the caudal edge of the trapezoid body to the rostra1 edge of the inferior olive. Dorsoventrally, the area extended for about 1 mm dorsal to the pyramid. Mediolaterally, most sites were located around 0.5 mm lateral to the midline. On the other hand, locomotion-inducing sites outside of these boundaries were observed in some animals. For example, low threshold locomotion could be induced in some cases following stimulation along the midline dorsal to the MED. Also, in a few cases, stimulation of an area dorsal to the MED and 0.5-l .O mm lateral to the midline led to locomotion. These locations produced locomotion in only a few cases, however, when evident, stimulation failed to produce replicable stepping. Examination of the histological material revealed that the above distribution of locomotion-inducing sites, the region herein termed the MED, appeared to be located in and around the area of the nucleus reticularis magnocellularis pars beta (23). Some sites were found to be located more ventrally in the area of the nucleus reticularis magnocellularis pars alpha. Other sites were found slightly more dorsally in the ventral part of the nucleus reticularis gigantocellularis. Locomotion was not evident following stimula-
Injection Sites Only one penetration per MED was made. At the end of each experiment, an appropriate volume of Fast Green (0.30.7 ~1) was injected through the cannula at 1 kl/min. Most animals (n = 29) were sacrificed with an overdose of barbiturate and perfused transcardially with fixative and sucrose for NADPH diaphorase histochemistry (44). Frozen sections were cut (40 km) sagittally and the extent of Fast Green spread assessed by overhead projection of wet sections. Drawings of the extent of the Fast Green spread from wet sections later were superimposed on drawings of the same sections stained for NADPH diaphorase. Because the incubation needed for such labeling tends to dissolve the Fast Green except in spots, use of dried sections would tend to
MEDIOVENTRAL
MEDULLA-INDUCED
511
LOCOMOTION
tion. Stepping typically was elicited after 5-20 set of stimulation as the current amplitude was increased to threshold. Locomotion could be maintained for prolonged periods (up to 5 min). As the current amplitude was increased, the step cycle became increasingly rigid leading to “goose-stepping,” especially in the forelimbs. With increasing current such alternation quickly shifted to a trot and a gallop, leading to rigidity beyond 60-80 kA. The threshold for inducing locomotion following stimulation of the MED was 23.62 13.1 p,A (mean and S.D.). Stimulation of the MED did not require high frequencies to maintain stepping. Unlike the MLR, which requires pulse frequencies of 20-60 Hz, the MED could be activated using frequencies as low as 5 Hz. Similar characteristics were reported for the MED in the cat (9). The present experiments did not attempt to survey the entire medulla for locomotion-inducing sites. Areas possibly equivalent to the pontobulbar locomotor strip (PLS) (33), and the dorsal and ventral tegmental fields (21), were not tested in the rat. Rather, this study was limited to the area of the MED found to lead to labeling of MLR neurons after injections of retrograde tracers ( 12). Chemically Induced Locomotion
PIG. 1. (A) Sag&al view of the rat brainstem (midbrain-pons-medulla) at lateral level L 0.4 showing the locations of the central gray (CG), inferior olive (IO), pyramidal tract (PT), nucleus of the sixth cranial nerve (VI) and the level of transection (T). The vertical lines indicate several representative rostrocaudal stereotaxic penetrations using a semimicroelectrode and the effects obtained following stimulation at various depths. The stars denote low threshold- (
